Currently, the treatment of every case of methicillin resistant Staphylococcus aureus (MRSA) infection costs the UK National Health Service £9000 (Wendin R: The economics of patient safety: can you afford not to be safe, Marsh Europe S.A. Report commissioned by The Royal College of Physicians, London; 2008), which amounts to more than £45 m per year.
Although there has been investment of £50 m to deep clean the hospitals, it has been reported that there has been a small increase in the number of infection cases. This may be due to the fact that there are particular target areas in a hospital environment where reoccurrence of MRSA and MSSA (methicillin susceptible Staphylococcus aureus) infections are more likely.
In addition, MRSA related deaths came to 1200 in 2008 (Office for National Statistics. Health indicators. Health Stat Q. 2008; 37:7.) and whilst this represents a drop when compared to previous years, a disproportionate number are nosocomial and demand the use of expensive antibiotics to combat. Despite this apparent levelling-off in terms of infection rate, it is vitally important to maintain vigilance and develop an in ‘use’ or point of care surveillance technology that allows for continued monitoring, decreased burden on the Health Service providers but also improvements in patient care.
The existing technique of swabbing and culturing is costly, time-consuming and slow to give results. This delay between swabbing and obtaining a positive or negative result undoubtedly has a human and financial cost that is, for the moment, difficult to quantify.
The magnitude of the health threat posed by Staphylococcus aureus (MRSA and MSSA) has not gone unnoticed by public health agencies and governments (Mackenzie, F. M, Struelens, M. J. Towner, K. J, Gould, I. M; Clin. Microbiol. Infect., 2005, 11, 937-954). An increase in the number of instances of community acquired (Panton Valentine Leukocidin positive) and hospital acquired MRSA and MSSA has led many agencies to consider the current control strategies to be ineffective. Carrier screening, surveillance and molecular typing all form the tools for integrated MRSA/MSSA control strategies. However, the weakest link in this strategy is the screening and detection. Whilst the current techniques are reliable, they are costly and time-consuming.
Speed of diagnosis, detection and identification is critical in the management of an infected patient. Traditionally, diagnosis and detection uses two main approaches; the first attempts to identify the pathogen at the site of infection, whilst the second aims to determine the most appropriate treatment with the aim of reducing morbidity and mortality (Caroline Atardo Genco and Lee Wetzler; Neisseria—Molecular Mechanisms of Pathogenesis. Caister Academic Press. 2010). Currently the most common identification techniques either rely on biochemical profiling as seen in the API style tests or alternatively using molecular techniques such as PCR or DGGE amongst others. Most commonly a combination of techniques is used (Sintchenko, V. Magrabi, F. Tipper, S; Medical Informatics and the Internet in Medicine, 2007, 32, 3, 225-240).
These tests vary in terms of completion times and in the levels of precision. The time taken can vary from a few hours up to 18 hours after initial pure culture and even longer in some cases. It is also well understood that certain phenotypic markers might be altered under the influence of differing environmental conditions. On occasions, antimicrobial susceptibility profiles are also used to aid in diagnosis which requires pure culture. In such situations bacterial identification is most likely to be achieved around 48 hours after initial sample reception, which can hamper patient treatment and have potential implications in terms of infection control.
Commonly, detection methods for these pathogens involve culture in liquid or on solid media. These techniques, including a more recent chromogenic cefatoxin based agar medium, typically detect MRSA and MSSA within 20 to 48 hours especially when the initial diagnosis is presumptive as seen with non-chromogenic detection methodologies. Rapid MRSA/MSSA detection is possible using PCR and hybridisation assays. However, whilst the microbiology can be performed within 1.5 to 6 hours, sampling and transport to an appropriately equipped laboratory means in reality the best turn-around time is 24 hours after admission of a patient. This assumes the testing can be performed 7 days per week 24 hours per day. Recent trials of conventional culture methods and PCR techniques have shown there is a lack of demonstrable benefit in the case of PCR based evaluation (Harbarth, S. Fankhauser, C. Schrenzel, J; JAMA, 2008, 299, 1149-1157; Jeyaratnam, D. Whitty, C. J. Phillips, K; British Medical Journal, 2008, 336, 927-930).
Another technique, is a variant on the tube coagulase test and is predicated on a tripeptide coupled to a coumarin fluorophore, first synthesised in 1977 (Ford, M. Perry, J. D. Robson, I. Morgan, S. Holliday, M. G. Orr, K. E. Gould, F. K; Journal of Hospital Infection, 1999, 41, 2, 133-135; Morita, T. Katcv, H Sadaaki, I. Takada, K. Kimura, T. Sakakibara, S; J. Biochem. 1977, 82, 1495-1498).
This tripeptide mimics fibrogen and is cleaved by the coagulase expressed by S. aureus (MRSA and MSSA). Loss of the tripeptide results in a change in UV absorption of the coumarin. The sensitivity of this dye is limited and requires cell culture prior to analysis. This is based, in no small part, on the low extinction coefficient of the coumarin fluorophore.
There are three broad groups of approaches towards testing for MRSA; conventional culture, immunocapture and molecular detection. The procedure for conventional culture is time-consuming, involves a sample swab being obtained from the patient/surface, sterile transport to a microbiology lab where it can be used to inoculate a chromogenic agar plate, followed by incubation. Results are not usually available until 24 to 48 hours after the initial swab. With regard to immunocapture methods, methods such as ELISA can be used, this involves either sample culture or polymerised chain reaction (PCR) replication of the sample swab to be carried out before results can be obtained. Single swabs can be processed in approximately 6 hours, however this time drops drastically when running multiple samples. This approach can also be very expensive, requiring a dedicated PCR capable microbiology laboratory.
There are also a number of commercial kits that allow for the detection of pre-formed staphylococcal toxins, however in many cases sample culture and purification is required prior to testing, making use of these kits limited at best. An example of these kits, which is often carried out within small laboratories, is the latex agglutination kit, allowing a result to be obtained in approximately 20 to 24 hours, however there are a number of known problems with the general applicability of these tests.
Rhodamine exhibits a high extinction coefficient and pronounced shift in absorption/emission characteristics in lactone or zwitterionic forms. An existing example for the detection of generic serine proteases is the fluorogenic substrate Ala-Pro-Pro-Cresyl Violet, which is specifically cleaved by dipeptidyl peptidase IV, allowing confocal imaging of DPPIV transfected Jurkat cells (Boonacker, E. Elferink, S. Bardai, S. Fleischer, B, Van Noorden, C; The Journal of Histochemistry and Cytochemistry, 2003, 51, 7, 959-968).
EP 2 036 897 discloses stable rhodamine labelled Phosphoramidites and synthesis supports which can be used as a labelling reagent for labelling of oligonucleotides during oligonucleotide synthesis.
WO 03/099780 discloses enzyme assays, and more particularly to protease assays using luminogenic protease substrates that include a rhodamine-based luminophore.
There is a need for rapid diagnosis of infectious agents such as MRSA and MSSA, especially in terms of point of care technologies. Indeed many health bodies are demanding active surveillance for MRSA and MSSA.
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